The tubulysins are members of a new class of natural products isolated from myxobacterial species (F. Sasse, et al., J. Antibiot. 2000, 53, 879-885). As cytoskeleton interacting agents, the tubulysins are mitotic poisons that inhibit tubulin polymerization and lead to cell cycle arrest and apoptosis (H. Steinmetz, et al., Chem. Int. Ed. 2004, 43, 4888-4892; M. Khalil, et al., ChemBioChem. 2006, 7, 678-683; G. Kaur, et al., Biochem. J. 2006, 396, 235-242). Tubulysins are extremely potent cytotoxic molecules, exceeding the cell growth inhibition of any clinically relevant traditional chemotherapeutic e.g. epothilones, paclitaxel, and vinblastine. Furthermore, they are potent against multidrug resistant cell lines (A. Dömling, et al., Mol. Diversity 2005, 9, 141-147). These compounds show high cytotoxicity tested against a panel of cancer cell lines with IC50 values in the low picomolar range; thus, they are of interest as potential anticancer therapeutics.
Tubulysins are described herein. Structurally, tubulysins often include linear tetrapeptoid backbones, including illustrative compounds having formula T

and pharmaceutically acceptable salts thereof;
wherein
Ar1 is optionally substituted aryl;
R1 is hydrogen, alkyl, arylalkyl or a pro-drug forming group;
R2 is selected from the group consisting of optionally substituted alkyl and optionally substituted cycloalkyl;
R4 is optionally substituted alkyl or optionally substituted cycloalkyl;
R3 is optionally substituted alkyl;
R5 and R6 are each independently selected from the group consisting of optionally substituted alkyl and optionally substituted cycloalkyl;
R7 is optionally substituted alkyl; and
n is 1, 2, 3, or 4.
Another illustrative group of tubulysins described herein are more particularly comprised of one or more non-naturally occurring or hydrophobic amino acid segments, such as N-methyl pipecolic acid (Mep), isoleucine (Ile),
and analogs and derivative of each of the foregoing. A common feature in the molecular architecture of the more potent natural occurring tubulysins is the acid and/or base sensitive N-acyloxymethyl substituent (or a N,O-acetal of formaldehyde) represented by R2-C(O) in the formula (T).
Another illustrative group of tubulysins described herein are those having formula 1.
Structures of several natural tubulysins
TubulysinRAR2AOHCH2CH(CH3)2BOHCH2CH2CH3COHCH2CH3DHCH2CH(CH3)2EHCH2CH2CH3FHCH2CH3GOHCH═C(CH3)2HHCH3IOHCH3
A total synthesis of tubulysin D possessing C-terminal tubuphenylalanine (RA═H) (H. Peltier, et al., J. Am. Chem. Soc. 2006, 128, 16018-16019) has been reported. Recently, a modified synthetic protocol toward the synthesis of tubulysin B (RA═OH) (O. Pando, et at., Org. Lett. 2009, 11, 5567-5569) has been reported. However, attempts to follow the published procedures to provide larger quantities of tubulysins were unsuccessful, being hampered in part by low yields, difficult to remove impurities, the need for expensive chromatographic steps, and/or the lack of reproducibility of several steps. The interest in using tubulysins for anticancer therapeutics accents the need for reliable and efficient processes for preparing tubulysins, and analogs and derivatives thereof. Described herein are improved processes for making natural tubulysins, or analogs or derivatives thereof, including compounds of formula (T) and formula (1).
In one illustrative embodiment of the invention, processes for preparing natural tubulysins, or analogs or derivatives thereof, including compounds of formula (T) and formula (1) are described herein. The processes include one or more steps described herein. In another embodiment, a process is described for preparing a compound of formula B, wherein R5 and R6 are as described in the various embodiments herein, such as each being independently selected from optionally substituted alkyl or optionally substituted cycloalkyl; and R8 is C1-C6 n-alkyl; wherein the process comprises the step of treating a compound of formula A with a silylating agent, such as triethylsilyl chloride, and a base, such as imidazole in an aprotic solvent.
It is to be understood that R5 and R6 may each include conventional protection groups on the optional substituents.
In another embodiment, a process is described for preparing a compound of formula C, wherein R5 and R6 are as described in the various embodiments herein, such as each being independently selected from optionally substituted alkyl or optionally substituted cycloalkyl; R8 is C1-C6 n-alkyl; and R2 is as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; wherein the process comprises the step of treating a compound of formula B with a base and a compound of the formula ClCH2OC(O)R2 in an aprotic solvent at a temperature below ambient temperature, such as in the range from about −78° C. to about 0° C.; wherein the molar ratio of the compound of the formula ClCH2OC(O)R2 to the compound of formula B from about 1 to about 1.5.
It is to be understood that R2, R5 and R6 may each include conventional protection groups on the optional substituents.
In another embodiment, a process is described for preparing a compound of formula D, wherein R5 and R6 are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R8 is C1-C6 n-alkyl; R2 is as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; and R7 is optionally substituted alkyl; wherein the process comprises the steps of
a) preparing a compound of formula (E1) where X1 is a leaving group from a compound of formula E; and
b) treating a compound of formula C under reducing conditions in the presence of the compound of formula E1.
It is to be understood that R2, R5, R6, and R7 may each include conventional protection groups on the optional substituents.
In another embodiment, a process is described for preparing a compound of formula F, wherein R5 and R6 are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R2 is as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; and R7 is optionally substituted alkyl; wherein the process comprises the step of treating compound D with a hydrolase enzyme.
It is to be understood that R2, R5, R6, and R7 may each include conventional protection groups on the optional substituents.
In another embodiment, a process is described for preparing a compound of formula G, wherein R5 and R6 are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R2 is as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; and R7 is optionally substituted alkyl; wherein the process comprises the step of treating the silyl ether of compound F with a non-basic fluoride containing reagent.
It is to be understood that R2, R5, R6, and R7 may each include conventional protection groups on the optional substituents.
In another embodiment, a process is described for preparing a compound of formula H, wherein R5 and R6 are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R2 and R4 are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; and R7 is optionally substituted alkyl; wherein the process comprises the step of treating a compound of formula G with an acylating agent of formula R4C(O)X2, where X2 is a leaving group.
It is to be understood that R2, R4, R5, R6, and R7 may each include conventional protection groups on the optional substituents.
In another embodiment, a process is described for preparing a tubulysin of formula (T), wherein Ar1 is optionally substituted aryl; R1 is hydrogen, optionally substituted alkyl, optionally substituted arylalkyl or a pro-drug forming group; R5 and R6 are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R3 is optionally substituted alkyl; R2 and R4 are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; and R7 is optionally substituted alkyl; wherein the process comprises the step of forming an active ester intermediate from a compound of formula H; and reacting the active ester intermediate with a compound of the formula I to give a compound of the formula T.
It is to be understood that Ar1, R1, R2, R4, R5, R6, and R7 may each include conventional protection groups on the optional substituents.